The present invention relates generally to molecular biology, and particularly to a new class of conjugate ligands based on hyperglycosylated cytokines.
Cytokines are soluble proteins which mediate the hormonal regulation of various cells and tissues. Cytokines deliver a signal to target cells by binding to integral membrane proteins or complexes of proteins, known as receptors. Cytokine receptors contain an extracellular ligand binding region and an intracellular region activated by cytokine binding, which delivers a signal to other intracellular components of the cell. The interaction of cytokines with the extracellular regions of cytokine receptors is highly specific.
Lymphokines are cytokines which regulate and coordinate the activities of various cell types in the immune system. Different immune cells express different populations of lymphokine receptors. For example, T cells, which are involved in the cellular immune response, express varying numbers of interleukin-2 (IL-2) receptors on their cell membranes, depending upon their state of activation. By enumerating the number of cells expressing IL-2 receptors in a particular plasma sample and the average number of receptors per cell, a sensitive assessment can be made of the extent of T-cell activation. Other classes of immune cells express other receptors, for example, receptors for IL-4, IL-3 and IL-1. If the appropriate technology were available, analysis of cytokine receptor expression could be employed as a sensitive tool to enumerate, image or isolate cells in various cell populations or tissue samples. This detection technology could form the basis for new avenues of diagnosis and therapy. For example, certain leukemias are characterized by expression of GM-CSF receptors on the leukemic cells. Such leukemias could be diagnosed and typed by flow cytometry if a reagent capable of detecting GM-CSF receptors were available.
Currently, counting and typing of immune and other circulating blood cells can be conducted by flow cytometry, for example, fluorescence-based cell sorting, wherein laser activation of fluorescent labeling dyes is employed to distinguish cells for separation in an electrostatic field. Flow cytometric apparatus and techniques are well developed, and have been reviewed by Parks and Herzenberg, Meth. Enzymol. 108:197 (1984) and Cambier and Monroe, Meth. Enzymol. 103:227 (1983). Cells may be labeled for flow cytometry using specific antibody for particular membrane proteins or receptors. In direct immunofluorescence studies, specific antibody is directly conjugated to a fluorescent dye moiety, for example, fluorescein isothiocyanate. In indirect immunofluorescence methods, an unlabeled specific antibody is detected using a labeled antibody which binds the Fc region of the unlabeled antibody.
The use of flow cytometric techniques to detect cytokine receptors is limited by the lack of specific antibodies for most receptors, which is due to the lack of purified receptor (or cell lines expressing high levels of receptor) for immunization. With a few exceptions, e.g., IL-2 receptor (p55 or Tac antigen), preparations of cytokine receptors sufficient to raise specific antibody are not available, due to the low abundance of many receptors on known cell types. For these reasons, biochemical studies of polypeptide hormone receptors have often been limited to indirect methods such as .sup.125 l-ligand crosslinking, due to the combined problems of low receptor abundance and the lack of specific antibody or other molecular probes.
An alternative approach to cytokine receptor detection by flow cytometry using antireceptor antibodies involves labeling the cytokine itself for use as a fluorescence reagent. For example, Shirakawa et al., J. Immunol. 138:4243 (1987) disclose labeling of human IL-1.alpha. with fluorescein isothiocyanate (FITC) to study the expression of IL-1 receptors on human peripheral T cells. Yamasaki et al., Science 241:825 (1988) describe flow cytometric analysis of cells based upon IL-6 receptor expression, using recombinant IL-6 conjugated to biotin, which was then labeled using FITC-avidin. This technique can also be used to purify receptors, based upon the high affinity (Kd 10.sup.-15 M) binding of biotinylated polypeptides to avidin and streptavidin for receptor affinity chromatography. One difficulty with this approach is the need to produce a biotinylated ligand which retains the ability to bind to its specific cell surface receptor. The most common approach has been to couple the N-hydroxysuccinimide ester of biotin to primary amines on the protein backbone, e.g., as disclosed by Yamasaki et al. However, if primary amino groups are critical to the receptor-binding function of the ligand, this approach is not feasible. One alternative approach to protein biotinylation involves conjugation to oligosaccharide moieties, which has been demonstrated with immunoglobulins. However, recombinant proteins produced in the most common bacterial expression systems (e.g., E. coli) lack glycosylation, and recombinant proteins produced in other systems are glycosylated only to a small extent or to an unpredictably heterogeneous extent.
Another problem in receptor detection by fluorescence-based flow cytometry is low levels of receptor expression. In order to discriminate cells on the basis of receptor expression where receptors are sparsely expressed, for example, at levels of 15-100 receptors per cell, it is necessary to employ a detection reagent which can be labeled with a sufficient number of dye moieties to permit detection of receptor-bearing cells above background levels of fluorescence. To detect receptors which are expressed at levels of 500-1000 receptors per cell requires a reagent with multiple dye residues per ligand. Conventional methods of cytokine labeling, for example, direct FITC-conjugation and FITC-avidin-biotin conjugation, are incapable of providing cytokine reagents with a sufficient number of fluorescein residues to detect many classes of receptors, for example, human GM-CSF and IL-3 receptors. To address these problems, new types of cytokine reagents are needed.
The present invention provides an improved class of cytokine reagents for use in flow cytometry and other receptor-based cell separation techniques in which functional groups, for example, biotin groups, are conjugated to oligosaccharide moieties. The improvement of the present invention resides in the use of recombinant hyperglycosylated cytokines, which are produced in yeast. Yeast are capable of expressing protein having higher levels of O-linked and N-linked mannose (glycosylation) than mammalian cells. The resulting "halo" of polysaccharide on the hyperglycosylated cytokine, which can account for as much as 70% of the weight of the molecule, provides a useful substrate for conjugation of multiple biotin or other linker moieties. Hyperglycosylated cytokines can be labeled to very high specific fluorescence levels without deleteriously affecting the receptor binding characteristics of the conjugate ligand. Hyperglycosylated cytokine conjugates can be employed not only in fluorescence-activated flow cytometry, but also in other analysis, imaging or separation techniques.